Project Summary Parathyroid hormone (PTH) maintains mineral ion homeostasis by interacting with the PTH/PTHrP receptor to induce transcriptional and post-transcriptional modifications in target tissues. PTH stimulates resorption of the mineral matrix in bone, and increases production of active vitamin D, calcium reabsorption, and phosphate excretion in the kidneys. Despite the central role of PTH as a regulatory hormone, many of the downstream intracellular target molecules that orchestrate PTH signaling events have yet to be identified. Characterization of these pathways will expand current knowledge of the mechanisms governing mineral homeostasis, and suggest new therapeutic strategies to counteract or restore PTH action in skeletal and renal disease. Recently, salt- inducible kinases (SIKs) were identified as intracellular mediators of PTH signaling in osteocytes, and small- molecule inhibitors of SIKs were found to mimic PTH action on bone in vivo. The objective of this proposal is to explore whether analogous SIK-dependent PTH signaling pathways are active in the kidney. Aim 1 of this proposal will explore the molecular mechanisms through which PTH/SIK signaling regulates CYP27B1 expression. In renal cells, human kidney organoids, and mice, both PTH and the SIK inhibitor YKL-05-099 increase expression of CYP27B1, the enzyme that converts inactive vitamin D to its active form. In renal cells, PTH treatment results in decreased phosphorylation of SIK3 substrate CRTC2. The remaining intermediaries of the PTH/SIK/CYP27B1 pathway will be characterized using in vitro experiments targeting molecules upstream and downstream of SIK in renal cells and organoids. Chromatin Immunoprecipitation of CRTC family members will identify the regulatory regions necessary for CYP27B1 transcriptional activation by PTH/SIK. Aim 2 will define the role of PTH/SIK signaling in renal phosphate reabsorption. Mice treated with PTH or YKL-05-099 have decreased serum phosphate levels, and YKL-05-099 treatment decreases phosphate transporter Npt2a localization to the renal brush border membrane. These mice will be tested to confirm that SIK inhibition induces phosphaturia in vivo. Three SIK family members will be knocked out in renal epithelial cells and their effect on phosphate uptake assessed. Aim 3 will characterize the phenotype of mice lacking renal SIK1 and SIK3, and explore the therapeutic potential of small molecule SIK inhibitors in CKD-MBD. The experiments described here explore a novel role for SIKs in regulating PTH signaling in the kidney. In addition, mouse models developed for this project will help to shed light on the crucial bone/kidney regulatory axis that controls mineral homeostasis and bone health. This project also offers a novel application for SIK inhibitors, a class of drug currently studied for their application in autoimmune and other inflammatory disorders, as treatment in later stages of chronic kidney disease (CKD) and other disorders of mineral ion homeostasis that are unresponsive to PTH.